Biomedical Engineering Reference
In-Depth Information
upstream of Smo, robotnikinin is a useful tool for probing earlier steps in the Shh
signaling pathway that were not previously accessible, creating new opportunities for
the development of anticancer drugs.
18.3.2
Inhibitor of the Secretory Pathway
The secretory pathway is a fundamental process for shuttling synthesized proteins
from the rough endoplasmic reticulum (rough ER) through the Golgi apparatus and
the trans-Golgi network (TGN) to the plasma membrane or a specific organelle [56].
Previous studies have established fundamental concepts relating to the biogenesis of
organelles and the mechanisms of vesicle formation and fusion. However, detailed
regulatory mechanisms are not fully understood in many important secretory-related
pathways [57]. Small-molecule modulators of the secretory pathway are highly useful
tools for studying these complicated biological processes, as the rapid movement of
organelles and intracellular traffic present a challenge for traditional genetic and
biochemical methods [7b,58].
The Shair and Kirchhausen groups have discovered an inhibitor, secramine, that
perturbs traffic out of the Golgi apparatus (anterograde transport) in the secretory
pathway through the use of a DOS library (Figure 18.2) [7]. The DOS library was
generated via a biomimetic strategy inspired by the biosynthesis of the natural product
galanthamine. An image-based phenotypic screening was developed to measure the
inhibition of traffic out of the Golgi by monitoring the localization of the indicator
vesicular stomatitis virus glycoprotein (VSVG). In this assay, the VSVG ts045 mutant
strain is retained in the ER at 40
◦
C due to reversible misfolding. When the temperature
is decreased to 32
◦
C, the protein redistributes into the Golgi complex and then into the
cell membrane. This process can be observed by automated fluorescence microscopy
when the strain is tagged with enhanced green fluorescent protein (EGFP) [59]. In
this study, secramine was identified as a potent inhibitor of VSVG
ts
-EGFP transport
from the DOS library [7].
A natural product that is also known to delay VSVG
ts
-EGFP transport from the
Golgi apparatus, cytochalasin B, is known to prevent actin polymerization [60]. Thus,
for target identification purposes, secramine was tested in an actin polymerization
inhibition assay. In this assay,
Xenopus laevis
cytoplasmic egg extract was used and
actin polymerization was monitored by the increase in fluorescence caused by the
polymerization of actin-pyrene. The dose-dependent inhibitory effects of secramine
on the rate of actin polymerization suggested that the target of secramine also exists
within the actin polymerization process. Actin polymerization is activated by PIP2
liposome stimulation of Cdc42 (a member of Rho family of small GTPases) via
its effector proteins, Taco-1 and N-WASP, which further recruit the actin-binding
Arp2/3 complex [7b]. When actin polymerization was activated directly by the
N-WASP VCA domain, secramine was found to be ineffective, which suggested
that secramine interferes with upstream processes, PIP2 liposome-stimulated Cdc42
activation, and/or Cdc42 effectors of activation. Secramine was next examined to
determine if it could affect the binding outcome between Cdc42 in the active, GTP-
bound state and another one of its effectors, PAK1. GST-PBD, a recombinant peptide
